A technical innovation in a structural biology field has deepened understanding of relationship between a structure of a protein and a function thereof, and has led to development of an innovative drug based on the structure of the protein. In an X-ray crystallographic structure analysis of the protein, crystallization of the protein is an essential step. Crystallization takes place through a process in which a crystal nucleus is formed in a supersaturated solution, and a process in which the crystal nucleus grows to a crystal.
An obstacle to the structural analysis resides in the crystallization step. Upon performing crystallization of a protein of which crystallization conditions are unknown, search for the crystallization conditions first becomes essential. The search for protein crystallization conditions contains a first screening and a second screening. The first screening contains a step of searching for physical conditions and chemical conditions for the purpose of determining conditions, by an extensive search for conditions of crystallization of a target protein, under which the target protein starts crystallization. In this step, for example, protein concentration, kind and a concentration of precipitant, kind of a buffer solution and pH thereof, and the like effective to crystallization are assessed to find out conditions allowing the crystallization to start. On the other hand, the second screening contains a step of searching for elaborated physical conditions and chemical conditions for the purpose of determining final conditions for crystallization of the target protein by modifying the crystallization start conditions selected through the first screening. Modification of the crystallization start conditions in the second screening is performed by partially changing the physical conditions and the chemical conditions constituting the crystallization start conditions or by selectively adding new physical conditions and chemical conditions that are not included in the crystallization start conditions. For example, finally optimum crystallization conditions, specifically, conditions for obtaining a single crystal having a larger possible size with a high quality are found out by finely adjusting conditions such as the concentration of the precipitant and the pH of the buffer solution, or by using an additive or a surfactant that is not used in the first screening, and also by finely adjusting the kind, the concentration or the like thereof. In any of these screenings, a trial-and-error examination of conditions is required for a limitless number of combinations of physical conditions and chemical conditions according to the target protein. For example, the second screening is performed through many kinds of examinations for conditions, such as kind of precipitant, pH of a solution, concentration of a protein solution, and temperature thereof. Crystals obtained after determining the crystallization conditions are subjected to a structural analysis by X-ray irradiation. Before irradiation with X-rays, the crystals may be occasionally immersed into an antifreeze containing a cryoprotectant. Moreover, when omission of the immersion step is desired, protein crystallization is tried using a precipitant solution containing the cryoprotectant in the first screening or the second screening. In any case, each screening is repeated until a desired crystal is obtained.
However, a protein is not generally active in forming crystal nuclei, and therefore hard to cause crystallization. In addition thereto, conditions of crystallization of a new protein cannot be anticipated at all, and new crystallization conditions cannot be anticipated even to a protein in which crystallization conditions are known, either. Under such a situation, the conventional first screening sometimes did not result in formation of crystals or even crystalline precipitates, and could not determine crystallization start conditions. Depending on the protein crystallization conditions, presence or absence of crystal nucleation is changed by a slight change of setting conditions, such as a solvent and temperature. Therefore, the conventional second screening that is the modification of the crystallization start conditions as selected in the first screening sometimes yielded no crystal. Control of protein crystal nucleation by the modification of setting conditions such as the solvent or the temperature is difficult. Therefore, the conventional second screening inclined to result in no crystal formation at a desired size and in the number of pieces. Moreover, the conventional second screening resulted in no crystal formation under conditions referred to as a metastable region in which supersaturation conditions are less than the crystallization start conditions, and in which no crystal nucleation takes place but crystal growth potentially takes place. When protein crystals are formed in the metastable region, the crystal inclines to have a sufficiently high quality, as is well known in the art. The cryoprotectant suppresses the protein crystal nucleation. Therefore, use of a reagent containing the cryoprotectant in the first screening and the second screening inclined to cause no protein crystallization. As described above, the conventional screening system inclined to yield no objective crystals or waste the protein being a precious sample after all even with an examination of conditions in which a load is high. Accordingly, a development has been required for a simple, economical and highly reliable method for searching for protein crystallization conditions.
In response thereto, an attempt was proposed for improving efficiency of protein crystallization by using a layered silicate. Patent Literature 1 discloses a screening chip for searching for protein crystallization conditions by using a layered silicate, in which the silicate is applied in the form of a film onto a support. However, the layered silicate described in Patent Literature 1 has a problem of not sufficiently promoting crystal nucleation of extensive proteins in screening on the assumption of searching for conditions of crystallization of various proteins. Moreover, the film is not a thin uniform film, and therefore is likely to be easily detached. Furthermore, the invention described in Patent Literature 1 requires a user of the screening chip to select an optimum type of the layered silicate and an amount of layered silicate application. Such an examination of conditions causes consumption of a large amount of protein solution. Moreover, the silicate is present in a part in which the screening chip and the protein solution are not in contact with each other. Therefore, extensive penetration of the protein solution inclines to decrease a degree of supersaturation and to cause protein denaturation.
Patent Literatures 2 and 3 disclose an agent for controlling protein crystal formation by using a layered silicate, and a method for controlling the formation using the same. However, the layered silicates described in Patent Literatures 2 and 3 also promotes only insufficient protein crystal nucleation in screening in a manner similar to the layered silicate described in Patent Literature 1. Moreover, the layered silicate described in Patent Literatures 2 and 3 has a weak surface negative charge and low water-swelling properties. Therefore, the layered silicate inclines to agglomerate and precipitate when the layered silica comes in contact with a screening reagent containing a precipitant at a high concentration.
Non-Patent Literature 1 discloses an agent for controlling lysozyme crystal formation containing a fluorine-containing layered silicate. Non-Patent Literature 1 discloses use of the layered silicate in lysozyme crystallization. However, no description is made on use of the layered silicate on the assumption of searching for conditions of crystallization of various proteins. Moreover, the layered silicate described in Non-Patent Literature 1 also insufficiently promotes the crystal nucleation of the protein in screening.
Non-Patent Literature 2 discloses an agent for controlling protein crystal formation that contains fluorine mica. This fluorine mica is synthesized by a solid-state reaction process, by using talc as a starting material, in the absence of moisture. However, the fluorine mica described in Non-Patent Literature 2 has no hydroxyl group. Therefore, crystal nucleation rate cannot be adjusted, and crystal nucleation is not promoted in some cases. Therefore, the fluorine mica is insufficient for an agent for searching for crystallization conditions. Moreover, the fluorine mica is non-swelling in water, and therefore inclines to agglomerate or precipitate in contact with a reagent containing a precipitant at a high concentration. Accordingly, use of the fluorine mica for a dispersion liquid needs agitation for uniformly dispersing the fluorine mica.
Non-Patent Literature 3 discloses an agent for controlling protein crystal formation that contains a mineral containing a layered silicate. The layered silicate constituting the mineral described in Non-Patent Literature 3 promotes protein crystal nucleation only insufficiently.
Non-Patent Literature 4 discloses an agent for controlling protein crystal formation that contains mica having an amino group. The mica described in Non-Patent Literature 4 forms a sheet-like material to be used for sealing the well in a hanging-drop process. This sheet-like material promotes protein crystal nucleation only insufficiently.